FIGURE 3.

Region comprising aa 16–24 contains a neutralization epitope. A, amino acid sequence of H77 HVR1 and the peptide expressed as a thioredoxin-peptide fusion protein that was used for rabbit immunization. B, HCV envelope proteins in lysates of 293T cells transfected with a panel of plasmids were coated onto microplates using GNA, and then the binding of the envelope proteins to IgG purified from six rabbits immunized with TRX-HVR1 peptide fusion proteins was measured by ELISA. The anti-HVR1 peptide IgG preparations were used at a concentration of 100 μg/ml. Conformation-dependent anti-E2 monoclonal antibody H53 (1:1,000 dilution) was used to assay E2 protein in cell lysate. The lysate of empty plasmid-transfected 293T cells was used as a negative control (Mock). C, similar inputs of various viral particles normalized for HCV E2 and HIV Gag proteins were preincubated with IgG (100 μg/ml) fractionated from sera collected from immunized or unimmunized rabbits at RT for 30 min before infection of Huh7.5 target cells. The infectivity of HCVpp was determined after 3 days as described above. VSV-GP was used as control. Results are the means ± S.D. of four independent experiments. D, total of 12 mice were immunized with HBsAg and H77 HVR1 fusion gene expression plasmids. Six mice (NS1–NS6) received HVR1-HBsAg expression plasmid and other six mice (SC1–SC6) received HBsAg-HVR1 expression plasmid. The mouse sera were collected at 2 weeks after the third immunization and were used as anti-HVR1 antibodies in ELISA. The data are the values for mouse sera at a dilution of 1:500. The mAb H53 diluted at 1:1,000 was used as control to assay envelope protein captured onto microplates. Mock means the lysate of 293T cells transfected with the empty vector.